The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent

Leah M Prentice,Teresa Ruiz De Algara,David G Huntsman,Xavier D'Anglemont De Tassigny,Margaret Sutcliffe,William H Colledge,Angela Burleigh,Steven Poon,Damian Yap,Pat Allard,Steven Mckinney,Gulisa Turashvili,Samuel Aj Aparicio,John Fee

doi:10.1186/1471-2164-12-209

Abstract

BackgroundHumans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods.ResultsWe show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.ConclusionsTaken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

Highlights

Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release gonadotropin releasing hormone (GnRH)
We compared quantile normalized probe intensity values from Affymetrix whole mouse exon array chips where hybridization was performed with wild-type (WT), Gpr54 knockout (GKO) or Kiss1 knockout (KKO) hypothalamic RNA
Affymetrix results were compared between genotypes, gene expression of all wild-type mice were grouped together and compared with all knockout mice grouped together (WT vs KO) or with Kiss1 knockout mice alone (WT vs KKO) or Gpr54 knockout mice alone (WT vs GKO)

Summary

Introduction

Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. In 2003 it was discovered that the kisspeptin activated GPR54 receptor is required for maturation and activity of the hypothalamic-pituitary-gonadal axis [1,2]. It has been established subsequently that kisspeptin (Kp) binding to GPR54 releases gonadotropin releasing hormone (GnRH) from the hypothalamus to stimulate the pituitary-gonadal axis in a multitude of species including humans [3,4,5,6,7,8,9,10,11,12,13,14,15]. Estrogen and testosterone have a negative feedback effect on Kp expression in the ARC that is regulated through the estrogen receptor alpha (ERa) or through the androgen receptor (AR) respectively [17,18,19,21,22]

Methods

Results

Discussion

Conclusion